After kidney ischemia/reperfusion (I/R) injury, monocytes home to the kidney and differentiate into activated macrophages. Whereas proinflammatory macrophages contribute to the initial kidney damage, an alternatively activated phenotype can promote normal renal repair. The microenvironment of the kidney during the repair phase mediates the transition of macrophage activation from a proinflammatory to a reparative phenotype. In this study, we show that macrophages isolated from murine kidneys during the tubular repair phase after I/R exhibit an alternative activation gene profile that differs from the canonical alternative activation induced by IL-4-stimulated STAT6 signaling. This unique activation profile can be reproduced in vitro by stimulation of bone marrow-derived macrophages with conditioned media from serum-starved mouse proximal tubule cells. Secreted tubular factors were found to activate macrophage STAT3 and STAT5 but not STAT6, leading to induction of the unique alternative activation pattern. Using STAT3-deficient bone marrow-derived macrophages and pharmacologic inhibition of STAT5, we found that tubular cell-mediated macrophage alternative activation is regulated by STAT5 activation. Both in vitro and after renal I/R, tubular cells expressed GM-CSF, a known STAT5 activator, and this pathway was required for in vitro alternative activation of macrophages by tubular cells. Furthermore, administration of a neutralizing antibody against GM-CSF after renal I/R attenuated kidney macrophage alternative activation and suppressed tubular proliferation. Taken together, these data show that tubular cells can instruct macrophage activation by secreting GM-CSF, leading to a unique macrophage reparative phenotype that supports tubular proliferation after sterile ischemic injury.

Conflict of interest: MLM developed thymus transplantation intellectual property, which has been licensed to Enzyvant Therapeutics. Both MLM and Duke University may benefit financially if the technology is commercially successful in the future.

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Neuronal calcium sensor-1 (NCS-1 Var1) is a calcium-binding protein expressed in most tissues. We examined a poorly characterized variant of NCS-1 (Var2), identified only in humans where the N-terminal 22 amino acid residues of native NCS-1(MGKSNSKLKPEVVEELTRKTY) were replaced with 4 different residues (MATI). Because alterations in the level of expression of NCS-1 Var1 and the expression of NCS-1 variants have been correlated with several neurological diseases, the relative expression and functional role of NCS-1 Var2 was examined. We found that NCS-1 Var2 mRNA levels are not found in mouse tissues and are expressed at levels ~1000-fold lower than NCS-1 Var1 in three different human cell lines (SHSY5Y, HEK293, MB231). Protein expression of both variants was only identified in cell lines overexpressing exogenous NCS-1 Var2. The calcium binding affinity is ~100 times weaker in purified NCS-1 Var2 than NCS-1 Var1. Because truncation of NCS-1 Var1 has been linked to functional changes in neurons, we determined whether the differing properties of the NCS-1 variants could potentially contribute to the altered cell function. In contrast to previous reports showing that overexpression of NCS-1 Var1 increases calcium-dependent processes, functional differences in cells overexpressing NCS-1 Var2 were undetectable in assays for cell growth, cell death and drug (paclitaxel) potency. Our results suggest that NCS-1 Var1 is the primary functional version of NCS-1.

Patients with 22q11.2 deletion syndrome and those with mutations in the Forkhead Box N1(FOXN1) transcription factor (Nude/SCID) can both present with a thymic hypoplasia that results in a severe T cell lymphopenia. In both clinical conditions, the thymic anlage fails to develop properly within the 3rdpharyngeal pouch during embryogenesis. We characterized the development of the thymus in mouse models of 22q11.2 deletion syndrome (22q11.2del) and a new set of mice with mutations in Foxn1that genocopied a SCID patient with novel compound heterozygous mutations in FOXN1. Both sets of mice develop hypoplastic thymic lobes. An analysis of thymopoiesis in embryos revealed distinct development problems. The hypoplastic thymii from the 22q11.2del mice were primarily sized restricted, with normal percentages of all thymocyte subsets apparent. This contrasted a severe deficiency of thymocytes due to the Foxn1mutations, which primarily affected the development and expansion of thymic epithelial cells. Comparative gene expression analyses of e13.5 fetal thymii revealed differentially regulated transcripts that define the basis of the hypoplasia. A dysregulated mesenchymal cell signature was apparent in the 22q11.2del model, which contrasted the epithelial transcript disruption due to the Foxn1mutations. These results suggest different strategies are necessary to correct the thymic tissue abnormalities in patients who present with thymic hypoplasias due to their congenital disorders.